In the biological and chemical sciences, there is often a need to separate particulate matter suspended in a solution. In a biological experiment, for example, the particles typically are cells, subcellular organelles and macromolecules, such as DNA fragments. A centrifuge is routinely used to perform the separation of such components from a solution.
The types of experiments that can be performed with a centrifuge are based primarily on three major sedimentation (fractionation) protocols, namely differential pelleting (differential centrifugation), rate-zonal density-gradient sedimentation and isopycnic density-gradient sedimentation. Basically, a centrifuge creates a centrifugal force field by spinning one or more tubes containing the solution to be separated, thus causing the suspended particles of interest to separate from the solution. The sedimentation rate of a particle is a function of such factors as the molecular weight and density of the particle, the centrifugal field acting upon the particle, and the viscosity and density of the solution in which the particle is suspended.
A differential pelleting experiment is primarily used for the sedimentation of particles according to size. The material to be fractionated is initially distributed uniformly throughout the sample solution. In a differential pelleting protocol, a centrifuge tube filled with the solution is spun to produce a centrifugal field which acts on the particles in the sample solution. Eventually, a pellet is formed at the bottom of the tube which is composed primarily of the larger particles present in the solution, but also includes a mixture of other smaller particles suspended in the solution.
A rate-zonal separation protocol is used to improve the efficiency of the fractionation by separating the particles according to size. Rate-zonal sedimentation of particles relies on the property that particles of different sizes (and therefore different masses) will migrate through a density-gradient at different rates when subjected to a centrifugal force field.
The technique involves layering a sample containing the components of interest onto the top of a liquid column which is stabilized by a density-gradient of an inert solute, typically sucrose. The maximum density of the gradient typically is less than the buoyant density of the components of interest, to allow migration of the components along the gradient. Upon centrifugation, the particles are driven down the gradient at a rate dependent upon factors including the mass and density of each particle, the density of the gradient, and the centrifugal forces acting upon each particle. Generally, the more massive particles will migrate at a faster rate than the lighter particles. With the passage of time, numerous "zones" or "bands" of particles having similar mass will form. As the centrifugation continues, the widths of the zones measured along the central axis of the centrifuge tube increase as well as the separation between bands. In addition, the zones themselves migrate toward the bottom of the tube, and eventually will coalesce at the bottom.
The third type of fractionation is an isopycnic density-gradient protocol, which relies on differences in the buoyant properties of the constituent particles dispersed in a high density solution as the basis for separation of the constituents. While centrifugation must proceed for a period of time sufficient to allow for banding, the protocol is an equilibrium technique in which separation essentially is independent of the time of centrifugation and of the size and shape of the constituents, although these parameters do determine the rate at which equilibrium is reached and the width of the zones formed at equilibrium.
There are two ways to prepare a solution for an isopycnic separation experiment. A solute having a pre-formed high density-gradient is provided, in which a sample containing the macromolecules is included. Subsequent centrifugation of the preparation will cause the macromolecules of the sample to migrate through the high density solute, forming bands at positions along the density-gradient corresponding to the buoyant density of each macromolecule. At each of these equilibrium positions, the buoyant force of the solute acting on a macromolecule is canceled by the opposing forces of the centrifugal field. Alternatively, the solution to be centrifuged may be prepared by mixing a solution of the macromolecules or particles of interest with a high density solute to give a uniform solution of both. In this case, the density-gradient forms during the centrifugation, with the particles forming bands along the resulting gradient as described.
Present centrifuge systems provide users with an interface for selecting the speed and duration of a centrifuge run. Additional parameters may be set, including a temperature setting for the run and the particular rotor to be used. Typically, a user will set up a centrifuge run first by deciding which of the three types of centrifuge protocols or experiments is appropriate for a given circumstance. Next, the user must determine the centrifugation speed and the run-time for the particular experiment and then set the centrifuge accordingly. Computing the run-speed and the run-time for an experiment depends upon a number of factors, such as the selected centrifuge protocol, the sedimentation rate of the particles of interest and knowledge of the parameters of the rotor to be used. In the case of density-gradient separations, namely the rate-zonal and isopycnic protocols, the gradient of the solute must be included in the computations as well.
A centrifuge is just one of a number of tools which the experimenter uses in solving the problem at hand, and so should be easy to use. Computing the operational run parameters for a centrifuge run and adjusting the centrifuge for the actual experiment generally do not relate to the problem being addressed. The experimenter thus is burdened with unnecessary detail, which tends to be distracting and therefore inefficient.
What is needed is a system and method which eliminate the extraneous steps of setting up a centrifuge for an experiment and which simplify setting up the centrifuge. The system and method, however, should also allow a user to directly manipulate the operational parameters of the centrifuge when unusual circumstances present themselves, requiring complete control over the centrifuge.